High-throughput phenotypic screening for functionally characterizing alt. exons

用于功能表征 alt 的高通量表型筛选。

• 批准号: 10702825
• 负责人: Thomas Gonatopoulos-Pournatzis
• 金额: $ 58.2万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10702825
• 关键词: A549; Alternative Splicing; Bar Codes; Biomedical Research; CRISPR screen; CRISPR/Cas technology; Cancer cell line; Cataloging; Cell Maintenance; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Consequentialism; Coupled; Custom; Data; Development; Disease; Dropout; Elements; Engineering; Ensure; Event; Exons; Future; Genes; Genetic; Genomics; Goals; Guide RNA; HCT116 Cells; Hematologic Neoplasms; High-Throughput Nucleotide Sequencing; Human; Hybrids; Individual; K-562; Libraries; Link; MDA MB 231; Malignant Neoplasms; Messenger RNA; Molecular; Names; Neurodevelopmental Disorder; Nuclear; Nuclear Localization Signal; Output; Pathway interactions; Phenotype; Physiology; Play; Polyadenylation; Process; Protein Isoforms; Protocols documentation; RNA; RNA Processing; RNA Splicing; RNA-Binding Proteins; Resolution; Role; System; Testing; Tissues; Transcript; United States National Institutes of Health; Vertebral column; Work; base; cancer cell; combinatorial; computational pipelines; design; embryonic stem cell; fitness; functional genomics; genomic locus; genomic platform; improved; insight; nuclease; pluripotency; polyadenylated messenger RNA; preservation; programs; screening; single-cell RNA sequencing; stem cells; tool; transcriptome; transcriptomics

Aim A In recent work performed at NCI/NIH, we have applied molecular engineering strategies to further boost the editing efficiency of CHyMErA. We have shown that addition of 6 nuclear localization signals at the C-terminus of Cas12a stimulates the editing efficiency of CHyMErA by promoting nuclear accumulation and stabilization of Cas12a nuclease. We are currently applying the improved CHyMErA screening platform to identify alternative exons that impact on cell fitness and proliferation in cancer cells. Comprehensive deletion of frame-preserving alternative exons will be performed using the CHyMErA screening platform across a panel of 7 diverse human cancer cell lines (A375, HCT116, MDA-MB-231, A549, Jurkat, K562, HAP1). These screens will enable the systematic cataloguing of the core and context-specific alternative fitness exons, along with providing insights into the functional complexity of the transcriptome. Aim B We have previously developed CHyMErA, a powerful and versatile tool for combinatorial perturbation of genomic loci, which is based on the expression of hybrid Cas9 and Cas12a gRNAs. This tool has been successfully applied for dropout and enrichment cell fitness screens. Compared to conventional single-cell transcriptomics, CRISPR screens coupled to scRNA-Seq require the capture of non-polyadenylated gRNAs in addition to the capture of mRNA in order to assign perturbations to consequential transcriptomic outputs. CHyMErA, in its current form, does not allow capturing of guide sequences using any scRNA-Seq platform. Therefore, we aim to transform CHyMErA to a perturbation tool that is amenable to capturing both the mRNA transcriptome and gRNAs at the single-cell level. To achieve this, we will introduce capture sequences in the middle of the tracrRNA of Cas9 and at the end of Cas12 gRNAs. These capture sequences will be recognized by customized RT barcoded primers during the first step of generating scRNA-Seq libraries. In parallel, barcoded polyT RT primers will be used to capture polyadenylated mRNA. Capture sequences have recently been used for a Cas9-based CRISPR platform and made available for use with the 3 Direct Capture 10x Genomics platform. We aim to use these capture sequences in our system but need to ensure that their incorporation does not impact the editing efficiency of CHyMErA. To test the suitability of scCHyMErA for combinatorial CRISPR perturbations coupled to scRNA-Seq, we will generate a small library targeting 450 genes using dual-targeting hgRNAs directing both Cas9 and the Cas12a to target the same gene. Two independent hgRNAs will be designed per gene resulting in 1,000 library elements (including controls) that will be cloned as a pool into a lentiviral backbone following protocols that we have mastered in the past. For our first screen we will focus on targeting genes encoding RNA binding proteins (RBPs) which regulate RNA processing events including alternative splicing and polyadenylation. These processes underlie important cell fate decisions and their disruption is linked to numerous diseases including hematological malignancies. The hgRNA library will be transduced into embryonic stem cells and subsequently subjected to single-cell RNA-Seq analysis profiling using standard 10x Genomics protocols.The transcriptomes will be sequenced on a NovaSeq 6000 S2 flow cell resulting in 4 billion reads equal to 50k reads per profiled cell and data will be analyzed using established computational pipelines. These data will not only establish scCHyMErA-Seq as a tool for CRISPR perturbation with scRNA-Seq readout but also may uncover genes and RNA-related pathways that play an important role in stem cell maintenance and pluripotency. Future screens will be focused on targeting tissue-regulated alternative cassette exons with scCHyMErA-Seq in order to uncover the postranscriptional splicing events that underlie developmental trajectories.

目的在NCI/NIH最近的工作中，我们应用了分子工程策略来进一步提高CHyMErA的编辑效率。我们已经证明，在Cas12a的C末端增加6个核定位信号，通过促进Cas12a核酸酶的核积累和稳定来刺激CHyMErA的编辑效率。我们目前正在应用改进的CHyMErA筛查平台来确定影响癌细胞适合性和增殖的替代外显子。使用CHyMErA筛选平台，将对7种不同的人类癌细胞系(A375、HCT116、MDA-MB-231、A549、Jurkat、K562、HAP1)进行全面的保留框架的替代外显子的删除。这些筛选将能够系统地对核心和特定背景的替代适应性外显子进行分类，并提供对转录组功能复杂性的见解。目的我们已经开发了基于杂合Cas9和Cas12a gRNAs表达的CHyMErA，这是一种强大而通用的基因组基因座组合扰动工具。该工具已成功应用于辍学和增富细胞健康筛查。与传统的单细胞转录切割技术相比，与scRNA-Seq连接的CRISPR筛选除了捕获mRNA外，还需要捕获非多腺化的gRNA，以便为随后的转录切割输出分配扰动。CHyMErA目前的形式不允许使用任何scRNA-Seq平台捕获引导序列。因此，我们的目标是将CHyMErA转化为一种扰动工具，能够在单细胞水平上同时捕获mRNA转录组和gRNAs。为了实现这一点，我们将在Cas9的trrRNA的中间和Cas12的gRNAs的末端引入捕获序列。在生成scRNA-Seq文库的第一步中，定制的RT条形码引物将识别这些捕获序列。同时，条形码Polyt RT引物将被用来捕获多腺苷化的mRNA。捕获序列最近已用于基于Cas9的CRISPR平台，并可用于3Direct Capture 10x基因组学平台。我们的目标是在我们的系统中使用这些捕获序列，但需要确保它们的合并不会影响CHyMErA的编辑效率。为了测试scCHyMErA对与scRNA-Seq耦合的组合CRISPR扰动的适用性，我们将使用双靶向hgRNAs生成一个针对450个基因的小文库，以指导Cas9和Cas12a针对同一基因。每个基因将设计两个独立的hgRNAs，从而产生1000个文库元件(包括对照)，这些文库元件将作为一个池克隆到慢病毒骨架中，遵循我们过去已经掌握的方案。在我们的第一个筛选中，我们将重点关注编码RNA结合蛋白(RBPs)的基因，这些基因调控RNA加工事件，包括选择性剪接和多聚腺苷化。这些过程是重要的细胞命运决定的基础，它们的破坏与包括血液系统恶性肿瘤在内的许多疾病有关。HgRNA文库将被转化为胚胎干细胞，随后使用标准的10倍基因组学协议进行单细胞RNA-Seq分析。转录本将在NovaSeq 6000 S2 Flow细胞上进行测序，结果是40亿个读数相当于每个分析细胞50k个读数，数据将使用已建立的计算管道进行分析。这些数据不仅将建立scCHyMErA-Seq作为CRISPR干扰scRNA-Seq读出的工具，还可能揭示在干细胞维持和多能性中发挥重要作用的基因和RNA相关途径。未来的筛选将重点放在利用scCHyMErA-Seq靶向组织调控的替代盒外显子，以揭示构成发育轨迹的转录后剪接事件。